Name: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles (Video) | JoVE Journal
Uploaded: 2012-08-31T12:00:00.000+00:00
Duration: 12 min 25 s
Description: Learn to reconstitute the MalFGK2 ABC transporter into nanodisc lipid particles using membrane scaffold proteins and gel filtration chromatography. This method enables functional studies of membrane proteins in a soluble, controlled environment.

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5 minutes

This work was supported by the Canadian Institute of Health Research. CSC was funded by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. FD is a Tier II Canada Research Chair.

Overall Reconstitution Process
 The reconstitution process starts by mixing the membrane scaffold protein (MSP) with the purified MalFGK2 complex in the presence of detergent-solubilized phospholipids. The step is followed by the slow removal of the detergent by an adsorbent polystyrene material called Bio-Beads or Amberlite (Figure 1). The auto-assembly process occurs most likely because of the apolar interactions between the hydrophobic phospholipids, the MalFGK2 complex and the surface of the MSP amphipathic protein. The final product is a discoid particle made of two molecules of MSP wrapping around the MalFGK2 complex. The particles are separated from the adducts and aggregates by ultra-centrifugation and analytical size-exclusion chromatography. The particles are characterized by native-gel electrophoresis and dynamic light scattering spectroscopy.
 1. Preparation of the Membrane Scaffold Protein, MSP
 <ol>
 <li>The his-tagged MSP (version MSP1D11) is produced from plasmid pMSP1D1 in E. coli BL21 (DE3) induced at OD600~0.5 with 0.5 mM isopropyl β-D-1-thiogalactopyranoside for 3 hr at 37 °C.</li>
 <li>The cells are harvested by centrifugation at 5,000 x g for 10 min at 4 °C and resuspended in TSG10 buffer containing 100 μM phenylmethanesulfonyl fluoride. </li>
 <li>The cells are lysed with a French press (3x) at 8,000 psi and the insoluble material is removed by centrifugation at 5,000 x g for 10 min at 4 °C. The soluble fraction containing the MSP is isolated by ultra-centrifugation at 125,000 x g for 45 min. </li>
 <li>The MSP is purified by nickel-chelating chromatography using ~1.5 ml Ni Sepharose HP in TSG10 buffer. Contaminants are washed away with TSG10 buffer containing 50 mM imidazole. The MSP is eluted with TSG10 buffer containing 600 mM imidazole. The purified protein is dialyzed in TSG10 buffer and stored in -70 °C at a protein concentration of ~10-15 mg/ml.</li>
 </ol>
 2. Preparation of the MalFGK2 Complex
 <ol>
 <li>The MalFGK2 complex, his-tagged at the C-terminus of MalK, is expressed from plasmid pBAD22-FGK in E. coli BL21 (DE3) and induced at OD600~0.5 with 0.2% L-arabinose for 3 hr at 37 °C.</li>
 <li>Following cell lysis and centrifugation as in step 1.3, the membrane pellet is resuspended in TSG20 buffer at a final concentration of 5 mg/ml. The material is solubilized with 1% w/v n-dodecyl-β-maltopyranoside (DDM) for 3 hr at 4 °C with gentle shaking.</li>
 <li>The insoluble material is removed by ultra-centrifugation as in step 1.3 and the supernatant containing the solubilized MalFGK2 complex is collected and purified as in step 1.4, but without dialysis.</li>
 <li>Further purification is achieved by gel filtration chromatography in TSGD buffer on a Superdex 200 HR 10/300 column at a flow rate of 0.5 ml/min.</li>
 </ol>
 3. Preparation of Phospholipids
 <ol>
 <li>An E. coli total lipid extract dissolved in chloroform is separated into 1,000 nmol aliquots in screw cap microfuge tubes. The solvent is evaporated under a gentle stream of nitrogen and dried further overnight in a vacuum desiccator.</li>
 <li>The lipid film is dissolved in TS buffer at 5 nM final concentration and vortexed vigorously and sonicated. The dissolved lipids will appear slightly opaque due to their general insolubility in aqueous TS buffer, but they should remain in suspension. DDM is added to a final concentration of 0.5% (~10 mM), at which the solution becomes clear.</li>
 <li>The lipid mixture is placed in a water bath and pulse sonicated for 5 times for ~5 sec. The lipid mixture is stored at 4 °C for a maximum of 1 week.</li>
 </ol>
 4. Preparation of Bio-Beads
 <ol>
 <li>Approximately 10-15 ml (dry volume) Bio-Beads is placed into a 50 ml tube.</li>
 <li>The beads are successively washed with 50 ml of 100% methanol, 95% ethanol, milliQ H2O, and finally TS buffer (twice each).</li>
 <li>The washed beads are stored at 4 °C in ~10 ml TS buffer.</li>
 </ol>
 5. Nanodisc Reconstitution
 <ol>
 <li>The MSP is diluted in TSGD buffer at the final concentration of ~7 mg/ml (~0.3 mM).</li>
 <li>~2 nmol of purified MalFGK2 complex are mixed at a protein:MSP:lipid ratio of 1:3:60 or 1:3:400 in TSGD buffer. The final concentration is 6 μM MalFGK2, 18 μM MSP and 360 μM lipids (1:3:60) or 2.4 mM lipids (1:3:400). The final volume is 300 μl. The final DDM concentration is 0.08% (~1.6 mM). The final concentration of glycerol depends on the amount of lipid added but remains around 5-10% v/v.</li>
 <li>~50 μl of Bio-Bead suspension is added to the tube and the mixture is incubated overnight on a rocking table at 4 °C.</li>
 <li>The beads are sedimented by gravity and the solution is pipetted through a narrow tip to avoid as much Bio-Beads as possible.</li>
 <li>Large precipitates are removed by ultra-centrifugation at 100,000 x g for 20 min. The discs are purified by gel filtration chromatography as in step 2.4 in TSG10 buffer. Fractions containing the discs are pooled and an aliquot should be re-injected on the same gel filtration column to test the stability of the preparation. </li>
 <li>The purified discs can stored at -70 °C for an extended period of time. Following thawing on ice, the discs should be subjected to ultra-centrifugation as in step 5.5 to remove potential precipitates. An aliquot should be analyzed by gel filtration chromatography to ensure that significant aggregation or precipitation did not occur during the thawing process. </li>
 </ol>
 6. Native Gel Electrophoresis
 <ol>
 <li>1 μM of nanodiscs (~0.2 mg/ml) are analyzed by native PAGE6, 7 (Tris-HCl pH 8.8, 4-12%) to assess the quality of the reconstitution (Figure 3A).</li>
 <li>The binding of MalE (1 μM) to the MalFGK2-complex reconstituted in nanodiscs is also assessed by native-PAGE (Figure 3A).</li>
 <li>After electrophoresis, the gel is stained with Coomassie blue for 10 min, and destained for ~1 hr.</li>
 </ol>
 7. Dynamic Light Scattering (DLS)
 <ol>
 <li>Nanodiscs are purified by gel filtration chromatography as in step 2.4 in TSG10 buffer using a Superdex 200 HR 10/300 column at a flow rate of 0.1 ml/min. Fractions containing nanodiscs are pooled and concentrated to ~10 mg/ml with an Amicon centrifugal filter.</li>
 <li>The sample is filtered twice (0.22 μm filter) prior to analysis by DLS using a DynaPro nanostar instrument (Wyatt Technology) in a 1 μl inner volume quartz cuvette. Data is fitted using the DYNAMICS software (Wyatt Technology) to estimate the diameter and molecular weight of the particles.</li>
 </ol>
 8. ATPase Measurements
 <ol>
 <li>The ATPase activity of MalFGK2-reconstituted nanodiscs is determined using a colorimetric assay8.</li>
 <li>1 μM of purified discs and 1 mM ATP are mixed together with increasing amounts of MalE and incubated at 37 °C for 20 min. The release of inorganic phosphate is measured at 660 nm.</li>
 <li>The amount of phosphate released is compared to a standard curve generated with a Phosphorous Standard solution.</li>
 </ol>
 9. Representative Results
 The nanodiscs are purified by gel filtration chromatography (Figure 2A, left). The chromatogram shows that the majority of the reconstituted discs (black trace) elute as a single peak, whereas discs made with excess lipids (red trace) elute in the void volume and as a series of broad peaks. The quality of the nanodiscs is further analyzed by native gel electrophoresis and dynamic light scattering spectroscopy (DLS). Properly reconstituted discs migrate as a sharp band on the gel whereas those reconstituted in the presence of an excess of lipids migrate as a smear (Figure 2A, right). Analysis by DLS shows that the disc population is homogeneous with an average diameter of 11.4 nm (Figure 2B). The reconstituted discs have an apparent molecular weight of 215 kDa based on the DLS approximation. Samples reconstituted in the presence of excess lipids display widely distributed radii around 100 nm, which is typical for non-homogeneous samples. 
 The quality of the MalFGK2 complex is assessed by native-gel electrophoresis and its activity by ATPase measurements (Figure 3). The maltose binding protein MalE binds with a high-affinity to the MalFGK2 transporter9, 10. Using non-denaturing gel electrophoresis, it is possible to detect a complex between MalE and MalFGK2 (Figure 3A). The stimulation of the MalK2 ATPase activity by MalE is shown in Figure 3B.
 <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="Nanodisc formation process using MSP and MalFGK₂ with gel filtration chromatography diagram." data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 Figure 1. Typical flowchart for the reconstitution protocol. 
 <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="Size exclusion chromatography and dynamic light scattering charts for protein aggregates and nanodiscs." data-altupdatedat="1787630408000" data-alt="Figure 2"> 
 Figure 2. Quality control of the nanodisc preparation. A. Gel filtration analysis (Superdex 200 HR 10/300 column) of the nanodiscs reconstituted at low lipid ratio (1/3/60; black trace) or high lipid ratio (1/3/400; red trace). Native gel electrophoresis of the same disc preparation. Molecular weight markers in kDa are indicated. B. Dynamic light scattering analysis of the same disc preparation. <a href="https://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
 <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="Electrophoresis diagram showing Nd-FGK2 MalE complex; ATP hydrolysis bar chart analysis." data-altupdatedat="1787630408000" data-alt="Figure 3"> 
 Figure 3. Analysis of the MalFGK2-nanodisc particles. A. Gel shift analysis of MalE incubated with increasing amounts of MalFGK2-nanodisc particles. B. ATPase activity of the MalFGK2-nanodisc particles as a function of MalE concentration.

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No conflicts of interest declared.

We describe a simple procedure for the reconstitution of the maltose transporter into nanodiscs. The transporter is ATPase active and the interaction with the soluble binding partner MalE can be recreated (Figure 3). The successful reconstitution of the transporter into nanodiscs open the way for additional biophysical and biochemical analysis. Of particular interest will be the systematic analysis the MalK ATPase and maltose transport activity in detergent, liposome and nanodiscs. ABC transporters c...

<table><tbody><tr><td>Amicon Ultra-4 50K centrifugal filter</td><td>Millipore</td><td>UFC805008</td><td>Follow manufacturer&#039;s protocol for proper use </td></tr><tr><td>Bio-Beads SM-2 Adsorbent</td><td>Bio-Rad</td><td>152-3920</td><td></td></tr><tr><td>E. &lt;em&gt;coli&lt;/em&gt; total lipids</td><td>Avanti Polar Lipids</td><td>100500C</td><td>Dissolved in chloroform, handle as appropriate for an organic solvent</td></tr><tr><td>Ni sepharose HP resin</td><td>GE Healthcare</td><td>17-5268-01</td><td></td></tr><tr><td>Phosphorous standard solution</td><td>Sigma-Aldrich</td><td>P3869</td><td></td></tr><tr><td>pMSP1D1</td><td>Addgene</td><td>20061</td><td></td></tr><tr><td>Superdex 200 HR 10/300</td><td>GE Healthcare</td><td>17-5172-01</td><td></td></tr><tr><td colspan="4">&lt;span class=&quot;jove_content&quot;&gt;&lt;strong&gt;Table I. &lt;/strong&gt; Specific reagents.&lt;/span&gt;</td></tr><tr><td colspan="4"> &lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Name &lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Composition&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;Comments&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;DDM stock&lt;/td&gt; &lt;td&gt;10% w/v DDM&lt;/td&gt; &lt;td&gt;Resuspend in milliQ water and store at -20 &amp;deg;C&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;MalFGK&lt;sub&gt;2&lt;/sub&gt; stock&lt;/td&gt; &lt;td&gt;1-2 mg/ml &lt;br/&gt; 50 mM Tris-HCl, pH7.9 &lt;br/&gt; 100 mM NaCl &lt;br/&gt; 10% v/v glycerol &lt;br/&gt; 0.03% w/v DDM&lt;/td&gt; &lt;td&gt;Store at -70 &amp;deg;C after purification&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;MSP stock&lt;/td&gt; &lt;td&gt;10-15 mg/ml &lt;br/&gt; 50 mM Tris-HCl, pH7.9 &lt;br/&gt; 100 mM NaCl &lt;br/&gt; 10% v/v glycerol&lt;/td&gt; &lt;td&gt;Store at -70 &amp;deg;C after purification in &amp;lt;1 ml aliquots and avoid excessive freeze/thaw cycles&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Phospholipid stock&lt;/td&gt; &lt;td&gt;5 nM &lt;em&gt;E. coli &lt;/em&gt;total lipids &lt;br/&gt; 0.5% w/v (10 mM) DDM &lt;br/&gt; 50 mM Tris-HCl, pH 7.9 &lt;br/&gt; 50 mM NaCl&lt;/td&gt; &lt;td&gt;Store at 4 &amp;deg;C for 1 week&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;TS buffer&lt;/td&gt; &lt;td&gt;50 mM Tris-HCl, pH 7.9 &lt;br/&gt; 50 mM NaCl&lt;/td&gt; &lt;td&gt;Store at 4 &amp;deg;C&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;TSG10 buffer&lt;/td&gt; &lt;td&gt;50 mM Tris-HCl, pH7.9 &lt;br/&gt; 100 mM NaCl &lt;br/&gt; 10% v/v glycerol&lt;/td&gt; &lt;td&gt;Store at 4 &amp;deg;C&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;TSG20 buffer&lt;/td&gt; &lt;td&gt;50 mM Tris-HCl, pH8 &lt;br/&gt; 100 mM NaCl &lt;br/&gt; 20% v/v glycerol&lt;/td&gt; &lt;td&gt;Store at 4 &amp;deg;C&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;TSGD buffer&lt;/td&gt; &lt;td&gt;50 mM Tris-HCl, pH7.9 &lt;br/&gt; 100 mM NaCl &lt;br/&gt; 10% v/v glycerol &lt;br/&gt; 0.03% w/v DDM&lt;/td&gt; &lt;td&gt;Store at 4 &amp;deg;C and add DDM just before use&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;3&quot;&gt;&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Table II. &lt;/strong&gt; Solution recipes.&lt;/p&gt;&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt; &lt;/table&gt; </td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

The overall goal of this procedure is to incorporate an A, b, C transporter. In this case, the maltose transporter malf GK two into soluble membrane nano disk particles. This is accomplished by first mixing the membrane scaffold protein MSP, with the transporter and the correct amount of lipids in detergent mycells.The second step is to add polystyrene beads and to gently rock the mixture overnight, the detergent is progressively absorbed onto the beads, allowing the formation of the nano disk particles. Next, the beads are removed by sedimentation. The final step is to separate the properly formed discs from aggregates and unassembled scaffold proteins by gel filtration chromatography.Finally, the quality of the discs is assessed by native gel electrophoresis or light scattering spectroscopy. The main advantage of this technique over other methods like liposomes, is that membrane proteins and discs are perfectly soluble in aqueous solution. In addition, the protein domains are now accessible from both side.Since now is no more membrane compartment Using membrane scaffold proteins of different lengths, I was able to trap and study the different ligament states of membrane proteins. Most importantly, this method can help answer key questions in the membrane proteomic field, such as the identification of novel binding partners of a, b, C transporters. In fact, the implications of this technique extend further toward therapy because it can help the discovery of activators or inhibitors of membrane proteins and receptors.Though this method seems simple, caution should be taken when choosing the correct protein lipids ratio. Generally, individuals new to this method while struggle in calculating the correct molder ratio or doing the correct control that ensure the quality of the preparation. Therefore, a visual demonstration of this method is very useful for a new student to learn the various steps and potential difficulties.To begin gro hexa histidine tagged MSP in e coli strain BL 21 DE three from the plasmid P MSSP 1D one induced protein production when the measured optical density at 600 nanometers is about 0.5 for three hours at 37 degrees Celsius, harvest the cells by centrifugation at 5, 000 Gs for 10 minutes at four degrees Celsius. Re suspend the pellet in TSG 10 buffer containing 100 micromolar PMSF lice the cells with a French press three times at 8, 000 PS.I remove the insoluble material by centrifugation at 5, 000 Gs for 10 minutes at four degrees Celsius. Then isolate the soluble fraction containing the MSP by ultracentrifugation at 125, 000 Gs for 45 minutes.Purify the MSP by nickel chelating chromatography using approximately 1.5 milliliters of nickel seros high performance in TSG 10 buffer. Wash away the contaminants with T SG 10 buffer containing 50 millimolar ILE elute. The MSP with TSG 10 buffer containing 600 millimolar I midaz after dialyzing.The purified protein in TSG 10 buffer MSP can be stored at minus 70 degrees Celsius and a protein concentration of approximately 10 to 15 milligrams per milliliter. The MALF GK two complex, his tagged at the C terminus of MAL K, is expressed from plasmid. PED 22 FG K grown in e coli strain BL 21 DE three induce protein production when the measured optical density at 600 nanometers is about 0.5 with 0.2%L Arabs for three hours at 37 degrees Celsius following cell lysis and centrifugation.As before, we suspend the membrane pellet in TSG 20 buffer to a final concentration of five milligrams per milliliter. Next, solubilize the material with 1%weight per volume GDM for three hours at four degrees Celsius with gentle shaking. Remove the insoluble material by ultracentrifugation as for purification of MAL FTK two.Then collect the supernatant containing the solubilized mal FTK two complex and purify again with nickel chelating chromatography. Further purify the complex by gel filtration chromatography in TSGD buffer on a SUEx 200, HR ten three hundred column at a flow rate of 0.5 milliliters per minute. To prepare phospholipids, an e coli total lipid extract dissolved in chloroform is separated into 1000 anomal aliquots in screw cap microfiche tubes evaporate the solvent under a gentle stream of nitrogen and drive further overnight in a vacuum.Desiccate next, dissolve the lipid film in TS buffer at five NMO final concentration and vortex vigorously then pulse sonicate five times for approximately five seconds. The dissolved lipids will appear slightly opaque due to their general insolubility in aqueous TS buffer, but they should remain in suspension. Add DDM to a final concentration of 0.5%At this point, the solution will become clear place lipid mixture in a water bath and puls sonicate five times for approximately five seconds.The lipid mixture can be stored at four degrees Celsius for a maximum of one week. To prepare the bio beads place approximately 10 to 15 milliliters of bio beads into a 50 milliliter tube. First, wash the beads with 50 milliliters of 100%methanol.Allow the beads to settle by gravity and then gently pour off the solution. Repeat the process once for methanol and then twice each for 95%Ethanol milli Q water and finally TS buffer. Begin nano discrete constitution by diluting MSP in TSGD buffer to the final concentration of approximately seven milligrams per milliliter or 0.3 millimolar.To ensure a successful experiment, it's important to choose the correct ratios of lipids and proteins to be mixed together. Mix approximately two M of purified malf GK two complex at a protein to MS P to lipid ratio of one to three to 60 in TSGD buffer for a final volume of 300 microliters here, a sample of excess lipid in the ratio of one to three to 400 is also prepared as a negative control at a 50 microliters of bio bead suspension to the tube and incubate the mixture overnight on a rocking table at four degrees Celsius the following day, sediment the beads by gravity. Once the beads have settled pipe at the solution through a narrow tip to avoid as much bio beads as possible.Remove large precipitates by ultracentrifugation at 100, 000 GS for 20 minutes. Proceed to purify the discs by gel filtration chromatography as performed earlier in TSG 10 buffer. Next, inject an Eloqua on the same gel filtration column to test the stability of the preparation.Then pool the fractions containing the discs. The quality of the preparation is assessed by native gel electrophoresis. Analyze one micromolar of nano discs to assess the quality of the reconstitution.Also assess the binding of mal E to the mal ftk two complex reconstituted in nano discs. After electrophoresis, stain the gel with kumasi blue for 10 minutes and detain for approximately one hour. To perform dynamic light scattering or DLS first purify nano discs by gel filtration chromatography as before, using a SUEx 200 HR ten three hundred column at a flow rate of 0.1 milliliters per minute.Pool fractions containing nano discs, and concentrate to approximately 10 milligrams per milliliter with an AmCon centrifugal filter. Then filter the sample twice. Perform analysis by DLS using a dynapro nano star instrument in a one microliter inner volume quartz vete fit data.Using the dynamic software to estimate the diameter and molecular weight of the particles, determine the A TPAs activity of mal FTK two reconstituted nano discs using a chole metric assay. First mix one micromolar of purified discs and one millimolar a TP with increasing amounts of mal e. Incubate the samples at 37 degrees Celsius for 20 minutes.Then measure the release of inorganic phosphate at 660 nanometers. Compare the amount of phosphate released to a standard curve generated with a phosphorus standard solution. The nano discs are purified by gel filtration chromatography.The chromatogram shows that the majority of the reconstituted discs elute as a single peak, whereas discs made with excess lipids elute in the void volume, and as a series of broad peaks, the quality of the disc preparation is further analyzed by native gel electrophoresis discs reconstituted with a limited amount of lipids migrate as a sharp band on the native gel, whereas those reconstituted in the presence of an excess of lipids migrate as smears. Analysis by DLS shows that the disc population is homogeneous with an average radius of 5.0 nanometers. Based on the DLS approximation, the reconstituted discs have an apparent molecular weight of 215 kilodaltons samples reconstituted in the presence of excess lipids display widely distributed radii around 100 nanometers, which is typical for non homogeneous samples.The activity of the MAL FTK two complex is assessed by measuring the rate of a TP hydrolysis and the affinity for interacting partners such as the maltose binding protein, MAL E, the maltose binding protein. MAL E binds with a high affinity to the MAL FTK two transporter using non denaturing gel electrophoresis. It is possible to detect a complex between MAL E and MAL ftk two, the stimulation of the MAL KA TPA activity by MAL E is shown here as a function of MAL e concentration.The making of the nano disc is relatively simple, yet small deviations from optimal conditions can lead to irreversible protein aggregation once mastered, this technique can be done in less than two days. While attempting this procedure, it's important to remember the importance of choosing a correct protein lipid ratio because an excess amount of lipids will lead to production of large poly disperse liposome like particles. It is this crucial to carefully analyze the quality of the reconstituted material using techniques other than gel filtration, such as light scattering spectroscopy, analytical ultracentrifugation, or more simply native gel electrophoresis.Remember, the type of phospholipid incorporating the disc as well as the of membrane scaffold protein can contribute to the efficiency of the reconstitution. After watching this video, we hope you got a good understanding of how to generate soluble and homogenous nano discs containing your membrane protein of choice. Good luck with your experiments.

a-step-step-method-for-reconstitution-an-abc-transporter-into

Research

JoVE Journal

Biology

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Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

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Learn to reconstitute the MalFGK2 ABC transporter into nanodisc lipid particles using membrane scaffold proteins and gel filtration chromatography. This method enables functional studies of membrane proteins in a soluble, controlled environment.

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

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